Achieving the full potential of intraarterial, intravenous, or pulmonary gene therapy requires quantitative bioengineering analysis of extracellular and intracellular barriers. With respect to the many viral and non-viral vectors tested, the field of gene transfer has been hampered by strong inflammatory and immunological responses during gene transfer as well as poor efficiency of gene transfer. Often, the readministration of the same virus serotype has proven extremely difficult due to neutralizing antibodies. We will investigate novel cationic glucocorticoids that deliver viral vectors and develop these formulations for anti-bacterial and anti-inflammatory activity. The following specific aims are proposed: Aim 1: Optimization of cationic steroids for improved delivery of Adeno- associated virus (AAV) serotypes to mouse lung. Delivery to normal or Pseudomonas aeurginosa infected mouse lung will be tested. We will measure the anti-inflammatory and bactericidal activity of cationic steroids in conjunction with gene expression and improved targeting to airway epithelium. We will test the ability of cationic steroids to facilitate vector readministration of the same AAV serotype. Aim 2: Enhancing gene transfer through chemical and siRNA screening to elucidate mechanisms and barriers of AAV gene transfer. We have conducted siRNA screening of over 5000 genes to identify a number of genes whose knockdown leads to either enhanced or reduced transgene expression following viral transduction. Both chemical and siRNA screening will be directed at understanding intracellular limits to viral transduction as a means for enhancing transgene expression and reducing viral doses during therapy. The overall project is focused on reducing side effects related to gene delivery, improving efficacy during gene delivery (even in the presence of local infection), and facilitating readministration of AAV vector to eliminate the need for biomanufacturing of multiple serotypes.